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Triggered Sequestration with DNA Nanostructures: A New Drug Delivery Method

Triggered Sequestration with DNA Nanostructures: A New Drug Delivery Method. August 14, 2006 iGEM Week 10: Progress Report Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel. Overview. Project goal Last week’s progress Test AscI digest Microcon filtration

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Triggered Sequestration with DNA Nanostructures: A New Drug Delivery Method

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  1. Triggered Sequestration with DNA Nanostructures: A New Drug Delivery Method August 14, 2006 iGEM Week 10: Progress Report Tiffany Chan, Katherine Fifer, Valerie Lau, Matthew Meisel

  2. Overview • Project goal • Last week’s progress • Test AscI digest • Microcon filtration • [Mg2+] and [oligo] titration • This week’s goals

  3. Project goal • To create a scaffolded DNA nanostructure that protects a ligand from degradation • When the ligand is presented on the outside of the nanostructure, we expect there to be no protection

  4. AscI digest • The “attachment oligo” and the “oligo ligand,” with 15 complimentary bps, were briefly incubated at room temperature • The double-stranded construct was then treated with AscI under four different digestion conditions • Results: Digestion for 24 min with 500mU of AscI works well. 25 bp+ 10 bp+ 10 bp+ 25 bp+ milliunits AscI: 0 0 0 50 500 0 50 500 digestion time: 0 0 12 12 12 24 24 24 (minutes) 18% PA, 1.5hr @ 120V, SYBR gold staining

  5. Microcon filtration Image courtesy Millipore • Samples are pipetted into the Microcon filter and are successively diluted and centrifuged • Results: relatively good oligo removal, but poor nanostructure yields 1 kb+ unpurified successive flow-throughs flowthrough scaffold retentate retentate 2% agarose, 10mM MgCl2, 1hr @ 60V, EtBr

  6. [Mg], [oligos]: PEG experiments 2% agarose, 10mM MgCl2, 1hr @ 60V, EtBr • PEG titration performed for each set of folding conditions • Results: Oligos were not cleared in any of the 0-8%-final PEG precipitations pellet pellet etc… supern. supern.

  7. [Mg], [oligos]: Microcon and PEG 1 3 5 7 9 11 13 15 17 19 2 4 6 8 10 12 14 16 18 20 • Results: PEG seems to be more efficient at removing oligos • Nanostructures in PEG seem to be damaged, gel shifting

  8. Other filtration options Prespin Load sample Centrifuge (large molecules elute first) • Microcon • use more material—is yield loss constant? • use a detergent to loosen nanostructures from membrane (Triton X-100?) • Sepharose spin columns • large molecules elute first • homemade: Datta et al. (2003), Anal. Biochem., 317, p. 284. • Clonetech Image courtesy Clonetech

  9. This week’s goals • Continue filtration/purification trials • AscI digest of folded nanostructure

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